Abstract
According to the National Institutes of Health (NIH), it is estimated that 65% and 80% of microbial and chronic infections, respectively, are associated with biofilms. Biofilm is a mixture of extracellular polymeric substances (EPS) that encapsulates microbial cells and provides advantages to the bacteria by protecting them from antimicrobial treatment and shielding them from immune system detection. Ultimately, biofilm heavily contributes to chronic infection. Currently, antibiotics are the most common form of treatment for bacterial infection. However, the presence of biofilm can make cells up to a thousand times more resistant to antibiotics, rendering the treatment insufficient for biofilm-associated infection. This leads to combination antibiotic treatment or use of high doses for long periods of time, both of which have inconsistent results and are not very safe or sustainable for the patient. Pseudomonas aeruginosa is one of the most frequently occurring opportunistic pathogens and its robust biofilm forming ability is a major factor contributing to its widespread hospital occurrence. One of the main components in P. aeruginosa biofilm is the exopolysaccharide alginate, which is known to act as a protective barrier and enhance bacterial adherence. As a critical virulence factor that contributes to chronic infection, alginate is an obvious target among efforts to improve treatment of multi-drug resistant P. aeruginosa. Providing advantages such as high specificity, biocompatibility, lower resistance development, and ability to be engineered for specific application, enzymatic treatment shows great promise for targeting biofilm exopolysaccharides.
Our lab previously demonstrated that polysaccharide lyase (PL), Smlt1473, from Stenotrophomonas maltophilia clinical reference strain k279a can depolymerize a variety of substrates including alginate and hyaluronic acid (HA) in a pH dependent manner. In this work, we characterize the activity of Smlt1473 against alginate produced by various mucoid P. aeruginosa clinical isolates from the UVA Health System. Collectively, we show that Smlt1473 has potential as an antibiofilm agent against P. aeruginosa, but that concentration of the enzyme required for inhibition and degradation of alginate varies among isolates.
Secondly, we explored the ability of Smlt1473 to improve nucleic acid extraction from polysaccharide-rich samples. Specifically, high quality and quantity RNA is crucial for molecular techniques such as RT-qPCR and RNA-seq that are used for gene expression analysis. High polysaccharide content, present in biofilm-producing bacteria, hinders cell lysis, decreases RNA yield, and reduces sample purity which ultimately limits the reliability and accuracy of downstream analysis techniques. We demonstrate that the use of Smlt1473 to pre-process samples prior to RNA isolation is directly relevant to Pseudomonas spp. as we observe improvement in RNA quantity and quality when the enzyme is involved in the isolation process as compared to when it is not. Use of Smlt1473 for sample pre-processing prior to RNA isolation is also able to improve gene assignment without introducing significant changes in gene expression.
Additionally, we utilized a variety of approaches including nature guided mutagenesis, molecular dynamics simulations, and generation of a directed evolution library to further understand the structure-function relationship for Smlt1473. Each of these approaches provided insight into residues that are involved in enzyme activity and substrate specificity. Leveraging nature and computational simulations allows us to make predictions about residues involved in enzyme activity and substrate specificity with decreased experimental burden. Ultimately, this work facilitates more effective protein design of Smlt1473 for specific applications.
Although S. maltophilia, the strain where Smlt1473 was first isolated, is a significant respiratory pathogen, this multi-drug resistant organism is highly understudied among Gram-negative bacteria. Since pathogenic strains of S. maltophilia tend to have Smlt1473, but environmental strains do not, this suggests a potential role for Smlt1473 in pathogenesis. To better understand pathogenic mechanisms of S. maltophilia, this work investigates the biological role of Smlt1473 in S. maltophilia with a focus on nutrient acquisition and metabolism.
Taken together, this dissertation demonstrates the potential for Smlt1473 to serve as an antibiofilm agent against P. aeruginosa and suggests utility of the enzyme for improved RNA isolation from polysaccharide-rich samples. Additionally, this work better informs protein engineering of Smlt1473 for specific applications and investigates the biological role of the enzyme in S. maltophilia pathogenesis.
